The long term objective of the parent grant is to analyze the molecular and cellular mechanisms during development and regeneration of skin appendages. The specific objective of this revision is to develop hair formation assay so these principles can be applied toward regenerative medicine. Progress has been made in producing hair follicles using dissociated multi-potential dermal and epidermal stem cells. However, a couple gaps need to be filled before significant progress in clinical applications can be made. Recently, we have improved significantly the previous assays by Lichti and Stenn into a simpler hair forming assay with clinically proper appearance and potential for high throughput assay. Our general hypothesis is that multi-potent skin stem cells can be generated by molecular reprogramming, and that arrangement and patterning of hair primordia can be regulated by environmental conditions. With the newly developed "planar hair forming procedure", we are able to apply our expertise in feather pattern formation to regulate the number and size of hair primordia. We will also apply our recent work on macro-environment regulation of hair stem cells to improve the efficiency of hair formation. The simple assay also allows us to search for the molecular mechanisms of trans- differentiation and de-differentiation of ectodermal organs. The development of this technology will bring new hope to those who suffer from severe wounds and alopecia. PUBLIC HEALTH RELEVANCE: Currently, over 100,000 patients are treated for burn injury in the United States per year, and burns can cause lasting cosmetic and functional defects to visible areas on the skin. Here we propose to develop a novel planar hair forming protocol which integrates progress from our parent grant and others'hair research and may bring new hope to those who suffer from severe wounds and alopecia.